Fatty acid desaturases are critical regulatory enzymes of unsaturated fatty acid biosynthesis and catalyze the conversion of a single bond between two carbon atoms (Cxe2x80x94C) to a double bond (Cxe2x95x90C) in a fatty acyl chain. The resultant double bond is often referred to as an unsaturated bond. Eukaryotic fatty acid desaturases, typically, are iron-containing enzymes that catalyze the NAD-(P)H and O2-dependent introduction of double bonds into methylene-interrupted fatty acid chains. Examination of the deduced amino acid sequence from mammals, fungi, insects, higher plants and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 or 4)H, HX(2 or 3), and HX(2 or 3)HH. This motif is also present in the bacterial membrane enzymes alkaline hydroxylase (omega-hydroxylase) and xylene monooxygenase.
There are three types of eukaryotic fatty acid desaturases, acyl-CoA, acyl-ACP, and acyl-lipid desaturases (Ntambi et al., Biochem. and Biophys. Res. Com. 266:1-4, 1999). In plants and cyanobacteria, acyl-lipid desaturases catalyzing most desaturation reactions and introduce unsaturated bonds into fatty acids that are in a lipid-bound form. Acyl-ACP desaturases are present in the plastids of plant cells and insert a double bond into fatty acids that are bound to acyl carrier protein (ACP). In animals, yeast and fungal cells, Acyl-CoA introduce unsaturated bonds into fatty acids that are bound to coenzyme A (CoA). A gene cloned from this family is stearoyl-CoA desaturase and this gene has been identified in many organisms including mice, rats, humans, yeast, ovines, and hamsters.
Fatty acid desaturases can introduce an unsaturated bond at a specific position in a fatty acyl chain, for example, at the xcex946, xcex949, or xcex9412 position. Desaturases are typically integral membrane proteins induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded (Ozols, J. (1997) MBC Vol. 8 (11): 2281-2290). Unsaturated fatty acids can be formed from a variety of fatty acids including palmitate and stearate resulting in the formation of unsaturated fatty acids palmitoleate (16:1), and oleate (18:1).
In mammals, the rate limiting step in the biosynthesis of monounsaturated fatty acids is the insertion of an unsaturated bond by stearoyl-CoA desaturase (SCD) in the xcex949 position of the fatty acid. SCD preferentially catalyzes the synthesis of oleic acid. Oleate enriched low density lipoprotein (LDL) exhibits increased affinity for the vessel wall, and is therefore pro-atherogenic (Rudel, L. L. et al. (1997) J. Clin. Invest. 1:100(1):74-83). SCD involvement in generating atherogenic LDL variants and in regulating triglyceride synthesis is further supported by the finding that polyunsaturated fatty acids (PUFA), which protect against atherosclerosis, negatively regulate the expression of the SCD gene (Rudel, L L et al. (1995) Atheroscler. Thromb. Vasc. Biol. 15(12):2101-10; Ntambi, J M (1999) J. Lipid Res. 40(9):1549-58). Moreover, a mouse deficient for SCD exhibits significant reduction in triglycerides (Miyazaki, M. et al. (2000) J. Biol. Chem, in press).
Unsaturated fatty acids play an important role in normal and diseased organisms. For example, the degree of fatty acid unsaturation in cell membrane lipids determines membrane fluidity. Moreover, the production of monounsaturated fatty acids, which once complexed with lipoproteins such as LDL, show increased affinity for the vessels wall has profound implications for cardiovascular disorders caused by aberrant fatty acid metabolism. Examples of such disorders include atherosclerosis, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, among others.
The present invention is based, in part, on the discovery of a novel fatty acid desaturase, referred to herein as xe2x80x9c25934xe2x80x9d. The nucleotide sequence of a cDNA encoding 25934 is shown in SEQ ID NO:1, and the amino acid sequence of a 25934 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 25934 protein or polypeptide, e.g., a biologically active portion of the 25934 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 25934 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167, wherein the nucleic acid encodes a full length 25934 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 25934 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 25934 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 25934 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 25934-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 25934 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 25934 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 25934 mediated or related disorders, e.g., a cardiovascular disorder. In another embodiment, the invention provides 25934 polypeptides having a 25934 activity. Preferred polypeptides are 25934 proteins including at least one desaturase domain, and, preferably, having a 25934 activity, e.g., a 25934 activity as described herein.
In other embodiments, the invention provides 25934 polypeptides, e.g., a 25934 polypeptide having the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number 2167; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number 2167, wherein the nucleic acid encodes a full length 25934 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 25934 nucleic acid molecule described herein.
In a related aspect, the invention provides 25934 polypeptides or fragments operatively linked to non-25934 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 25934 polypeptides.
In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 25934 polypeptides or nucleic acids.
In still another aspect, the invention provides a process for modulating 25934 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment or prophylaxis of conditions related to activity or expression of the 25934 polypeptides or nucleic acids, such as cardiovascular, neurological, metabolic, reproductive (e.g., ovarian), renal and hepatic disorders.
Examples of cardiovascular disorders include e.g., atherosclerosis, thrombosis, heart failure, ischemic heart disease, angina pectoris, myocardial infarction, sudden cardiac death, hypertensive heart disease; non-coronary vessel disease, such as arteriolosclerosis, small vessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and chronic pulmonary disease; or a cardiovascular condition associated with interventional procedures (xe2x80x9cprocedural vascular traumaxe2x80x9d), such as restenosis following angioplasty, placement of a shunt, stet, stent, synthetic or natural excision grafts, indwelling catheter, valve or other implantable devices.
In a preferred embodiment, the cardiovascular disorder is caused by aberrant fatty acid metabolism. Examples of disorders involving aberrant fatty acid metabolism include, but are not limited to, atherosclerosis, arteriolosclerosis, hypertriglyceridemia, obesity, diabetes, hypercholesterolemia, xanthomatosis, and hyperlipidemia. Most preferable, the disorder is atherosclerosis.
In the cardiovascular applications, the agent is administered alone or in combination with a cholesterol lowering agent. Examples of cholesterol lowering agents include bile acid sequestering resins (e.g. colestipol hydrochloride or cholestyramine), fibric acid derivatives (e.g. clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g. troglitazone), or hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors (e.g. statins, such as fluvastatin sodium, lovastatin, pravastatin sodium, or simvastatin), an ApoAII-lowering agent, a VLDL lowering agent, an ApoAI-stimulating agent, as well as inhibitors of, nicotinic acid, niacin, or probucol. Preferred cholesterol lowering agents include inhibitors of HMG-CoA reductase (e.g., statins), nicotinic acid, and niacin.
The cholesterol lowering agent can be administered prior to, at the same time, or after administration of the agent, in single or multiple administration schedules. For example, the cholesterol lowering agent and the agents of the invention can be administered continually over a preselected period of time, or administered in a series of spaced doses, i.e., intermittently, for a period of time.
In a preferred embodiments, the agent, alone or in combination with the cholesterol lowering agent, inhibit (block or reduce) atherosclerotic lesion formation or development, e.g., so as to inhibit lipid accumulation, increase plaque stability or promote lesion regression.
In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) fatty acid metabolism, in a subject. The method includes administering to the subject an agent that modulates the activity or expression of a 25934 polypeptide or nucleic acid, in an amount effective to modulate the conversion of saturated fatty acids to unsaturated fatty acids, e.g., monounsaturated fatty acids.
In a preferred embodiment, the 25934 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 25934 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 200, 250, 300 or more contiguous amino acids of SEQ ID NO:2.
In a preferred embodiment, the 25934 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1 or 3. In other embodiments, the 25934 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or more contiguous nucleotides of SEQ ID NO:1 or 3.
In a preferred embodiment, the agent modulates, e.g., decreases or blocks, desaturase activity or expression. For example, the agent (e.g., a polyunsaturated fatty acid, or nicotinic acid or niacin) may negatively regulate 25934 protein expression.
In a preferred embodiment, the agent modulates (e.g., decreases or increases) the activity or expression of a 25934 polypeptide or nucleic acid such that modulation of one or more of: lipoprotein composition, lipid (e.g., triglyceride) levels, apolipoprotein B, total cholesterol, or lipoprotein (a), occurs. For example, the agent can decrease low density lipoprotein (LDL)-cholesterol levels, and/or lipid (e.g., triglyceride) levels. Preferably, the agent can also increase high-density lipoprotein (HDL)-cholesterol levels.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 25934 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial or natural product library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 25934 nucleic acid or a fragment thereof, or any combination thereof.
In a preferred embodiment, the subject is a patient undergoing a therapeutic or prophylactic protocol. Preferably, the subject is a human suffering from, or at risk of a cardiovascular disease, e.g., atherosclerosis, thrombosis, heart failure, ischemic heart disease, angina pectoris, myocardial infarction, sudden cardiac death, hypertensive heart disease; non-coronary vessel disease, such as arteriolosclerosis, small vessel disease, nephropathy, hypertriglyceridemia, obesity, diabetes, hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and chronic pulmonary disease; or a cardiovascular condition associated with interventional procedures (xe2x80x9cprocedural vascular traumaxe2x80x9d), such as restenosis following angioplasty, placement of a shunt, stet, stent, synthetic or natural excision grafts, indwelling catheter, valve or other implantable devices.
In a preferred embodiment, the subject is a human suffering from, or at risk of a disorder involving aberrant fatty acid metabolism. Examples of such disorders include, but are not limited to, atherosclerosis, arteriolosclerosis, hypertriglyceridemia, obesity, diabetes, hypercholesterolemia, xanthomatosis and hyperlipidemia. Most preferable, the disorder is atherosclerosis.
In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
In a preferred embodiment, the agent is administered alone or in combination with a cholesterol lowering agent. Examples of cholesterol lowering agents include bile acid sequestering resins (e.g. colestipol hydrochloride or cholestyramine), fibric acid derivatives (e.g. clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g. troglitazone), or hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors (e.g. statins, such as fluvastatin sodium, lovastatin, pravastatin sodium, or simvastatin), an ApoAII-lowering agent, a VLDL lowering agent, an ApoAI-stimulating agent, as well as inhibitors of, nicotinic acid, niacin, or probucol. Preferred cholesterol lowering agents include inhibitors of HMG-CoA reductase (e.g., statins), nicotinic acid, and niacin.
The cholesterol lowering agent can be administered prior to, at the same time, or after administration of the agent, in single or multiple administration schedules. For example, the cholesterol lowering agent and the agents of the invention can be administered continually over a preselected period of time, or administered in a series of spaced doses, i.e., intermittently, for a period of time.
In a preferred embodiment, the agent, alone or in combination with the cholesterol lowering agent, inhibits (blocks or reduces) atherosclerotic lesion formation or development, e.g., so as to inhibit lipid accumulation, increase plaque stability or promote lesion regression.
In a preferred embodiment, the agent, administered alone or in combination with the cholesterol lowering agent, results in a favorable plasma lipid profile (e.g., increased HDL and/or reduced LDL).
In yet another aspect, the invention features a method of treating or preventing a cardiovascular disorder (e.g., atherosclerosis), in a subject. The method includes administering to the subject an agent that modulates the activity or expression of a 25934 polypeptide or nucleic acid, in an amount effective to treat or prevent the cardiovascular disorder.
In a preferred embodiment, the 25934 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 25934 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 200, 250, 300 or more contiguous amino acids of SEQ ID NO:2.
In a preferred embodiment, the 25934 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1 or 3. In other embodiments, the 25934 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or more contiguous nucleotides of SEQ ID NO:1 or 3.
In a preferred embodiment, the agent modulates, e.g., decreases or blocks, desaturase activity or expression. For example, the agent (e.g., a polyunsaturated fatty acid, or nicotinic acid or niacin) may negatively regulate 25934 protein expression.
In a preferred embodiment, the agent modulates (e.g., decreases or increases) the activity or expression of a 25934 polypeptide or nucleic acid such that modulation of one or more of: lipoprotein composition, lipid (e.g., triglyceride) levels, apolipoprotein B, total cholesterol, or lipoprotein (a), occurs. For example, the agent can decrease low density lipoprotein (LDL)-cholesterol levels, and/or lipid (e.g., triglyceride) levels. Preferably, the agent can also increase high-density lipoprotein (HDL)-cholesterol levels.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 25934 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial or natural product library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 25934 nucleic acid or a fragment thereof, or any combination thereof.
In a preferred embodiment, the subject is a patient undergoing a therapeutic or prophylactic protocol. Preferably, the subject is a human suffering from, or at risk of a cardiovascular disease, e.g., atherosclerosis, thrombosis, heart failure, ischemic heart disease, angina pectoris, myocardial infarction, sudden cardiac death, hypertensive heart disease; non-coronary vessel disease, such as arteriolosclerosis, small vessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and chronic pulmonary disease; or a cardiovascular condition associated with interventional procedures (xe2x80x9cprocedural vascular traumaxe2x80x9d), such as restenosis following angioplasty, placement of a shunt, stet, stent, synthetic or natural excision grafts, indwelling catheter, valve or other implantable devices.
In a preferred embodiment, the subject is a human suffering from, or at risk of a disorder involving aberrant fatty acid metabolism. Examples of such disorders include, but are not limited to, atherosclerosis, arteriolosclerosis, hypertriglyceridemia, obesity, diabetes, hypercholesterolemia, xanthomatosis and hyperlipidemia. Most preferable, the disorder is atherosclerosis.
In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
In a preferred embodiment, the agent is administered alone or in combination with a cholesterol lowering agent. Examples of cholesterol lowering agents include bile acid sequestering resins (e.g. colestipol hydrochloride or cholestyramine), fibric acid derivatives (e.g. clofibrate, fenofibrate, or gemfibrozil), thiazolidenediones (e.g. troglitazone), or hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors (e.g. statins, such as fluvastatin sodium, lovastatin, pravastatin sodium, or simvastatin), an ApoAII-lowering agent, a VLDL lowering agent, an ApoAI-stimulating agent, as well as inhibitors of, nicotinic acid, niacin, or probucol. Preferred cholesterol lowering agents include inhibitors of HMG-CoA reductase (e.g., statins), nicotinic acid, and niacin.
The cholesterol lowering agent can be administered prior to, at the same time, or after administration of the agent in single or multiple administration schedules. For example, the cholesterol lowering agent and the agents of the invention can be administered continually over a preselected period of time, or administered in a series of spaced doses, i.e., intermittently, for a period of time as a therapeutic or preventative measure.
In a preferred embodiment, the agent, alone or in combination with the cholesterol lowering agent, inhibits (blocks or reduces) atherosclerotic lesion formation or development, e.g., so as to inhibit lipid accumulation, increase plaque stability or promote lesion regression.
In a preferred embodiment, the agent, administered alone or in combination with the cholesterol lowering agent, results in a favorable plasma lipid profile (e.g., increased HDL and/or reduced LDL).
The invention also features a method of diagnosing a disorder, e.g., a cardiovascular disorder (e.g., atherosclerosis), in a subject. The method includes evaluating the expression or activity of a 25934 nucleic acid or a 25934 polypeptide, such that, a difference in the level of 25934 nucleic acid or 25934 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder. Because 25934 is regulated by Niacin (a clinically used therapeutic), 25934 levels and/or activity may be considered a marker for Niacin activity and or efficacy.
In a preferred embodiment, the subject is a human.
In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, is obtained from the subject.
In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 25934 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 25934 nucleic acid or polypeptide.
In a preferred embodiment, the disorder is a cardiovascular disorder, e.g., a cardiovascular disorder as described herein.
In a preferred embodiment, the disorder is atherosclerosis.
The invention also provides assays for determining the activity of or the presence or absence of 25934 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In a further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 25934 polypeptide or nucleic acid molecule, including for disease diagnosis, predicting triglyceride levels, predicting response to Niacin, and predicting a response to triglyceride therapy.
In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity of a 25934 polypeptide, e.g., a 25934 polypeptide as described herein, or the expression of a 25934 nucleic acid, e.g., a 25934 nucleic acid as described herein, including contacting the 25934 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the polypeptide or nucleic acid. Such agents are useful for treating or preventing a 25934-mediated disorders, e.g., cardiovascular disorders (e.g., atherosclerosis) or metabolic disorders (e.g., obesity or diabetes).
In a preferred embodiment, the contacting step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, is obtained from the subject.
In a preferred embodiment, the contacting step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 25934 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 25934 nucleic acid or polypeptide.
In a preferred embodiment, the activity of the 25934 polypeptide is protein desaturase activity, e.g., catalyzing the conversion of saturated fatty acids to unsaturated fatty acids. In such embodiment, the 25934 polypeptide is contacted with a saturated fatty acid, e.g., oleic acid, palmitate and stearate.
In a preferred embodiment, the agent is an inhibitor (partial or complete inhibitor) of 25934 polypeptide activity or expression.
In a preferred embodiment, the agent modulates one or more of: lipoprotein composition, lipid (e.g., triglyceride) levels, LDL, HDL, apolipoprotein B, total cholesterol, or lipoprotein (a). For example, the agent can decrease low density lipoprotein (LDL)-cholesterol levels, and/or lipid (e.g., triglyceride) levels. Preferably, the agent can also increase high-density lipoprotein (HDL)-cholesterol levels.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, a triple helix molecule, or a 25934 nucleic acid, or any combination thereof.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.